This application seeks to understand mechanistic elements of the myosin motor of striated muscle and to determine how specific human mutations disrupt myosin function and lead to skeletal muscle disease or cardiomyopathy. We will employ a multidisciplinary approach that takes advantage of the powerful genetic tools available for Drosophila melanogaster along with a broad range of expertise that allows us to study myosin in an integrative manner, from its crystal structure and kinetic function through its effects upon muscle ultrastructure, fiber mechanical properties and locomotion. For Aim 1, we will build the first models of human distal arthrogryposis syndromes, which cause skeletal muscle contractures of varying severity. Using transgenic Drosophila, we will assess effects upon myosin structure, ATPase activity, in vitro motility, indirect flight and jump muscle ultrastructure, fiber mechanics and organism locomotion. We will test the hypothesis that distal arthrogryposis mutations cause abnormal interactions with nucleotides yielding enhanced ADP binding, reduced sliding velocity, slowed relaxation dynamics and hypercontraction and that the severity of the defects correlates with the severity of the human syndromes. For Aim 2, we will examine the mechanistic basis of myosin dysfunction caused by two hypertrophic cardiomyopathy mutations. We will construct organisms expressing point mutations that change the charge of the disease-causing residues and then test predicted suppressor mutations for functional rescue in vitro and in vivo. This will reveal interactions altered by the initial mutatins and provide direct insight into protein-protein interactions that are critical for myosin function. We will test the hypothesis that mutations in residues that cause human hypertrophic cardiomyopathy alter contacts between the strands of the central ?-sheet of myosin or interactions between the N-terminal domain of myosin and the lever arm, resulting in increases in ATPase activity, actin sliding velocity, fiber power levels and myofibrillar disarray. For Aim 3 we will examine the ability of the Drosophila heart to hypertrophy as a result of expressing myosin mutations known to cause human hypertrophic cardiomyopathy. This will be the first attempt to determine how the simple Drosophila heart tube, which is known to exhibit dilated, constricted or hypertrophic phenotypes, reacts to human contractile protein mutations that cause hypertrophy. We will monitor effects upon RNA transcription, calcium handling, ultrastructure and cardiac physiology and determine whether manipulating signaling pathways by gene knockdown or pharmacological intervention ameliorates observed defects. We will test the hypothesis that myosin mutations that cause human hypertrophic cardiomyopathy result in significant wall thickening, abnormal myofibrillar arrays, arrhythmias, disrupted calcium signaling, and transcript profiles that mimic the human condition, and that these pathologies can be suppressed by pharmacological treatment or genetic intervention. Overall our project will take advantage of an innovative experimental system to test significant questions regarding basic myosin function and disease.